Given this, we adapted the Qmci for use in Dutch-language countries and validated the Dutch version, the Qmci-D, against the Dutch translation of the Standardised Mini-Mental State Examination (SMMSE-D).Differentiating mild cognitive impairment (MCI) from dementia is important, as treatment options differ. There are few short (<5 min) but accurate screening tools that discriminate between MCI, normal cognition (NC) and dementia, in the Dutch language. The Quick Mild Cognitive Impairment . The Qmci-D and SMMSE-D were administered sequentially but randomly by the same trained rater, blind to the diagnosis.The Qmci-D was more sensitive than the SMMSE-D in discriminating MCI from dementia, with a significant difference in the area under the curve (AUC), 0.73 compared to 0.60 (p = 0.024), respectively, and in discriminating dementia from NC, with an AUC of 0.95 compared to 0.89 (p = 0.006). Both screening instruments discriminated MCI from NC with an AUC of 0.86 (Qmci-D) and 0.84 (SMMSE-D).The Qmci-D shows similar,(good) accuracy as the SMMSE-D in separating NC from MCI; greater,, accuracy differentiating MCI from dementia, and significantly greater accuracy in separating dementia from NC. Given its brevity and ease of administration, the Qmci-D seems a useful cognitive screen in a Dutch population. Further study with a suitably powered sample against more sensitive screens is now required.The Q The prevalence of mild cognitive impairment (MCI)  and dememci) screen was developed. Based upon the ABCS 135 [mci is more sensitive in differentiating MCI from NC than the SMMSE and ABCS 135 [mci has six subtests, orientation, registration, clock drawing, delayed recall, verbal fluency and logical memory. It is scored out of 100 points, can be administered and scored in less than 5 min and has excellent test-retest reliability [mci correlates highly with the Standardised ADAS-cog, Clinical Dementia Rating scale and the Lawton-Brody activities of daily living scale [To address these challenges, the Quick Mild Cognitive Impairment  and to compare its sensitivity and specificity in differentiating MCI from NC and dementia to the most widely used short cognitive screen in the Netherlands, the Dutch version of the SMMSE (SMMSE-D).The goal of the present study was to adapt the Qmci was performed with a combined qualitative and quantitative approach [mci was translated to Dutch by a health professional with a good understanding of English, whose primary language is Dutch. This Dutch version was reviewed by an expert panel of Dutch health professionals and researchers and a completed version of the Qmci-D was generated. A professional, native English language-speaking translator, without knowledge of the concepts behind the screening tool, performed the back-translation. The back-translation was then reviewed by the original developers of the Qmci screen, who approved the final version. The Qmci-D was pre-tested on participants with normal cognition before it was used in this study.The translation of the Qapproach . The oriConsecutive patients were recruited during a four month-period, between November 2013 and March 2014, from a geriatric outpatient department in a regional hospital, in the North of the Netherlands, where they were referred for the assessment of cognitive problems. Normal controls were recruited by convenience sampling among healthy participants without cognitive problems, who were accompanying the patients. A diagnosis of dementia  was made using DSM-IV  and NINCThe MCI and dementia groups underwent the same comprehensive review at baseline including neuropsychological assessment and Magnetic Resonance Imaging or Computerized Tomogram. The purpose and procedure of the research were explained in advance and all participants signed an informed consent before participation in the study. The Medical Ethical Committee of the University Medical Center Groningen evaluated the study and judged that it did not need ethical approval under Dutch law.a priori, to establish the sample size, using the WINPEPI software programme PAIRSetc [mci compared to the SMMSE [mci would be 85 % compared to approximately 65 % for the SMMSE, to differentiate MCI from NC. To detect a 20 % (medium to large effect size) difference in sensitivity and specificity, between the two tests, at a significance level of 0.05 and power of 80 %, 76-paired observations were required. The sample size needed to distinguish MCI from dementia was not estimated as a significant difference between the Qmci and the SMMSE would not be expected [A power calculation was performed PAIRSetc , 27. Bashe SMMSE , it was expected .mci-D  and SMMSE-D  on the same day, in a counterbalanced fashion, blind to the final diagnosis.Demographic data (age and gender) were collected during each visit to the geriatric department. Patients were classified by a consultant geriatrician and divided into three groups . A trained rater administered both the Qmci-D and SMMSE-D scores, between groups, were tested by one-way analysis of variance (ANOVA). Analysis of covariance (ANCOVA) was used to test differences between participant groups, controlling for participant characteristics such as age. Post hoc pair-wise comparisons were performed using the Tukey’s honest significant difference (HSD) test. A p-value < 0.05 was regarded as significant. Bootstrapped ROC curves were generated [mci-D and SMMSE-D [Data were analyzed using SPSS version 20.0 and the statistical programming language R. The Shapiro-Wilk test was used to test for normality. Differences in Qenerated  to analy SMMSE-D . Differe SMMSE-D .p < 0.001). The NC group had a mean Qmci-D score of 64 (SD 10.5) and a mean SMMSE-D score of 28 (SD 1.8). The mean Qmci-D score for the MCI group was 46 (SD 11.8) compared with 24 (SD 2.9) for the SMMSE-D, while the dementia group scored 34 (SD 15.8) for the Qmci-D and 22 (SD 4.4) for the SMMSE-D. These scores and demographic data are summarized in Table In total, 90 participants, 41 males (46 %) and 49 females (54 %), were included in this study. In this sample, 35 (39 %) had NC, 30 (33 %) had MCI and 25 (28 %) were diagnosed with dementia. The overall mean age of the sample was 72.9, standard deviation (SD) of 9.1 years. The NC group (mean age 68.7) was younger than the MCI (mean age 79.1) and dementia (mean age 79.2) groups . Post hoc pair-wise comparisons using the Tukey’s HSD test showed significant mean differences between groups (p < 0.05). The mean scores for the Qmci-D also differed significantly between the three groups; . The differences between groups are presented as boxplots in Fig. One-way ANCOVA, used to test for differences in SMMSE-D scores between the three groups , showed that the mean scores differed significantly across the three groups , compared to 0.89 (95 % CI 0.80–0.96) for the SMMSE-D . Both the Qmci-D and the SMMSE-D discriminated MCI from NC, with ANOVA post-hoc tests showing a significant mean difference between the MCI and NC groups (p < 0.001). The AUC of the Qmci-D, in discriminating MCI from NC, was marginally greater at 0.86 (95 % CI 0.77–0.95), compared to 0.84 (95 % CI 0.74–0.94) for the SMMSE-D . At the point estimate the Qmci-D had a sensitivity of 70 % and specificity of 94 % compared to a sensitivity of only 60 % and a similar specificity of 94 % for the SMMSE-D. As for the discrimination of MCI from dementia, ANOVA post-hoc tests, showed a significant mean difference (p < 0.001) between these participants with the Qmci-D. The difference between scores for the MCI and dementia groups, for the SMMSE-D (p = 1.02) was not significant. The score test for homogeneity of variances across groups in ANCOVA indicated that homogeneity for the Qmci is rejected. Heteroscedasticity-corrected SEs and Tests after ANCOVA [mci-D mean scores after correction for age, between MCI and NC as well as between MCI and dementia, were significantly different from zero. The ability of the Qmci-D, to discriminate MCI from dementia, was significantly greater (p = 0.024) than the SMMSE-D, AUC of 0.73 (95 % CI 0.59–0.85) compared to 0.60 (95 % CI 0.45–0.75), respectively , providing the optimal sensitivity and specificity, are presented in Fig. see Fig. , b. The see Fig. , d. Thisr ANCOVA  indicateSee Fig. , f. At tmci-D and SMMSE-D for differentiating MCI from mild dementia cases alone was unchanged at 0.62 and 0.54, p = 0.03, respectively.When moderate and severe dementia cases were removed from analysis, the AUC of the Qmci for use in Dutch-language countries and to explore its concurrent validity against the most commonly used short cognitive screen in Dutch, the SMMSE-D. The results show that the Qmci-D is more accurate than the SMMSE-D in differentiating dementia from NC. It had only fair accuracy (AUC 0.73) at differentiating MCI from dementia, although it was significantly more accurate than the SMMSE (AUC 0.60). In this study however, the SMMSE-D wasn’t able to discriminate MCI from dementia with a very poor sensitivity of 24 %, particularly when moderate to severe cases were excluded. Based upon this data it would appear that both instruments have limited ability to separate MCI from dementia. This is markedly different from the initial validation of the English language version of the Qmci against the SMMSE [mci showed significantly greater accuracy over the SMMSE. This discrepancy may relate to the small sample size, suggesting that this study was underpowered to show superiority of one instrument over the other. This said, the goal of this study was not to show superiority of a Dutch language version of the Qmci, the Qmci-D, rather it was to show the concurrent validity of the translation against a widely used screening instrument.The goals of this study were to adapt the Qhe SMMSE , which iThe strength of this study is the robust analysis with bootstrapped ROC curves and 95 % confidence intervals, to identify the discriminatory characteristics of both screening tools. This method provides more accurate results than non-bootstrapped methods, especially when analyzing smaller sample sizes. The 95 % confidence intervals obtained from the bootstrap and the asymptotic approach , were inmci [mci-D only with the SMMSE-D. This was because the SMMSE is the most widely used short cognitive screen [mci the comparator was the SMMSE, allowing direct comparison with the results of that study [. The authors also caution that screening for cognitive impairment continues to have challenges and in clinical practice [The study has limitations. First, the diagnosis of MCI was based on clinical criteria, which may have increased the heterogeneity of this group and led to some bias. However, no single gold standard criterion for MCI exists and there is still a lack of uniformity in the clinical diagnosis of MCI between studies . This samci . Patientmci . Frontote screen  and in tat study . The autat study , 36, theat study  and shorat study . The autpractice  it remaimci-D against the SMMSE-D. The data suggests that the Qmci-D, although statistically significantly more accurate than the SMMSE-D, is limited in its ability to differentiate MCI from dementia. The results also suggest that the accuracy of both instruments at distinguishing MCI from NC was good although the Qmci was more accurate than the SMMSE in separating dementia from NC in a Dutch speaking population. Given this, albeit limited analysis in a small sample, as well as its brevity and ease of administration [mci, the Qmci-D, appears useful as a short cognitive screen. Further research is now required to confirm these findings with a larger sample including other dementia subtypes and to compare the test to other cognitive screens including the MoCA and M-ACE.In conclusion, this study shows the concurrent validity of the Qstration –17, 40,


Silk fibroin (SF) is a protein with bulky hydrophobic domains and can be easily purified as sericin-free silk-based biomaterial. Silk fibroin modified chitosan nanoparticle (SF-CSNP), a biocompatible material, has been widely used as a potential drug delivery system. Our current investigation studied the bio-effects of the SF-CSNP uptake by liver cells. In this experiment, the characterizations of SF-CSNPs were measured by particle size analysis and protein assay. The average size of the SF-CSNP was 311.9 ± 10.7 nm, and the average zeta potential was +13.33 ± 0.3 mV. The SF coating on the SF-CSNP was 6.27 ± 0.17 μg/mL. Moreover, using proteomic approaches, several proteins involved in the ubiquitin proteasome pathway were identified by analysis of differential protein expressions of HepG2 cell uptake the SF-CSNP. Our experimental results have demonstrated that the SF-CSNP may be involved in liver cancer cell survival and proliferation. Bombyx mandarins and Bombyx mori, other moth genera such as Antheraea, Cricula, Samia and Gonometa, and numerous other insects. The primary structure of silk fibroin is characterized as natural block copolymers comprising hydrophobic blocks with a repetitive sequence of short side-chain amino acids such as glycine and alanine, as well as mainly consisting of the recurrent amino acid sequence (Gly–Ser–Gly–Ala–Gly–Ala)n. The raw state consists of two main proteins, sericin and fibroin, with a glue-like layer of sericin coating two singular filaments of fibroin called brins [Silk fibroin (SF) is an insoluble protein with bulky hydrophobic domains, which consists of layers of antiparallel β sheets, and can be easily purified as sericin-free silk-based biomaterials . It is eed brins ,3. Such ed brins . SF-baseed brins ,6,7,8, aed brins ,10,11,12ed brins . These pBiomaterials play important roles in regenerative medicine, tissue engineering and drug delivery . NanomedThe tumor microenvironment includes surrounding blood vessels, cells, signaling molecules, and the extracellular matrix (ECM) . Most tuThe silk fibroin modified chitosan nanoparticles (SF-CSNPs) have continued to gain more attention as drug delivery carriers because of their better stability, low toxicity, simple and mild preparation methods, and because they provide versatile routes of administration . The SF-et al., in 1994 [“Proteome” and “proteomics” are relatively new words, coined by Wilkins  in 1994 . The proIn this study, we investigated various methods to analyze and characterize the parameters that influence the uptake of cells on SF-CSNP. The CCL-13 and HepG2 cells served as cell models for the uptake of SF-CSNPs. For instance, using colorimetric techniques such as the lactate dehydrogenase (LDH) and Bromodeoxyuridine  assays are convenient methods and are typically applied to much of the current biomaterial research. Characterizations of SF-CSNPs were observed by particle size and zeta potential analysis. To evaluate early responses of CCL-13 and HepG2 cells to SF-CSNPs, a mass spectrometry-based proteomic profiling system was adopted for assessing characteristic proteins that were expressed due to the interactions of CCL-13 and HepG2 cells with SF-CSNPs. Through the investigation, various proteins that influence the early responses of CCL-13 and HepG2 cells on SF-CSNP were found; and, as far as we know, some have not yet been reported in the study of cell-nanoparticle interactions.2 functional groups of glucosamine units to NH3+ ion. After the SF adsorbed onto the CSNP surface, the zeta potential was neutralized by the functional groups (COO−) of SF, and the SF-CSNPs were formed by layers self-assembled on CSNPs. The zeta potential of CSNP was around 15.13 mV, so the negatively charged SF molecules promoted the self-assembly with positively charged CSNPs. The zeta potential of the SF-CSNPs decreased significantly by approximately 2 mV (around −11.9%). Thus, the electrostatic forces can promote the aggregation of SF molecules and consequently, it is expected that the SF may easily self-assemble on CSNP surfaces. The conjugation of surface-modified SF with CSNPs has shown an average zeta potential of 13.33 mV, which demonstrates that the surface of CSNPs was well modified with SF. The data is shown in In this study, the CSNP was used as the base material and the SF was adsorbed onto the surface of CSNP. The SF-CSNPs were washed and isolated by ultracentrifugation  and then re-suspended in distilled deionized water (1 mL) by manual shaking. The protein concentrations of the SF-CSNPs suspension were measured by Bio-Rad Bradford total protein assay kit  with a mean of 6.27 ± 0.17 μg/mL. The result of protein assay for the CSNP suspension was not detected. As determined by particle size and zeta potential analyzers, the average size of the SF-CSNP was 311.9 ± 10.7 nm with the morphology of a spherical micelle, and the average zeta potential was +13.33 ± 0.3 mV in phosphate-buffered saline. The particle size of SF-CSNP was larger than CSNP, due to the SF adsorbed onto the CSNP surface. The surface charge of nanoparticles, also called zeta potential, is a concept used to describe the electrokinetic properties of a colloidal particle under the influence of an applied electric field, which can influence the nanoparticle stability in suspension through the electrostatic repulsion between nanoparticles. A larger zeta potential value means the particles are more stable and less likely to form aggregates. In our study, the surface charge of CSNPs was positive, resulting from the protonation of NHin vitro uptake of SF-CSNPs was evaluated by fluorescence microscopy. The HepG2 cell was incubated with the growth medium containing SF-CSNPs at a concentration of 5 μg/mL for 12 h at 37 °C. As expected, no red fluorescence signals were detected in sections of the cells without SF-CSNPs . Compared with the control, the BrdU was upregulated in the groups treated with CSNPs and SF-CSNPs with significant increase . Those results indicate that no significant cytotoxicity was observed in the CSNPs and SF-CSNPs groups; and in addition, the CSNPs and SF-CSNPs showed improved cell growth and proliferation. Unlike metal or metal oxide nanoparticles, the CSNPs and SF-CSNPs were non-toxic. The cell growth and survival rate were increased with the higher concentration of CSNPs and SF-CSNPs. As shown in As shown in To investigate the effect of SF-CSNPs on normal and tumor cells of the liver, a proteomic approach, such as revised phase nano-ultra performance liquid chromatography electrospray ionization-tandem mass spectrometry (RP-nano-UPLC–ESI-MS/MS) analysis, was utilized to analyze cell lysates. The traditional methods use individual antibodies to evaluate the cell response to nanoparticles, but the proteomic approach can be used to analyze an enormous number of proteins simultaneously. In this study, HepG2 and CCL-13 cells were incubated with SF-CSNPs. After 12 h, the HepG2 and CCL-13 cells were lysed, and the cell lysates were digested by trypsin. The tryptic peptides generated were subsequently analyzed by RP-nano-UPLC–ESI-MS/MS, respectively. The fragmentation spectra obtained by the RP-nano-UPLC–ESI-MS/MS analysis in the gradient detection mode were compared with a non-redundant protein database using Mascot software. When a protein was identified by three or more unique peptides, no visual assessment of spectra was conducted and the protein was considered to be present in the sample.In this study, more than one hundred proteins were identified and most of these were identified at the minimal confidence level, which was only one unique peptide sequence matched. Experimental results reported a total of nine protein identifications with higher confidence levels (at least three unique peptide sequences matched), and exhibited significant differences between the SF-CSNPs treated HepG2 and CCL-13 cells. A summary of the protein identifications achieved is shown in In addition, there were five proteins identified in HepG2 cell lysate samples, which were involved in apoptosis, transcription, mitosis, cell division and cycle regulation. The five proteins with the molecular function of cell growth were positively identified as apoptotic chromatin condensation inducer in the nucleus , AT-rich interactive domain-containing protein 1A , Baculoviral IAP repeat-containing protein 6 , G2/mitotic-specific cyclin-B2 , and Centrosome-associated protein CEP250 . The protein-protein interaction pathways were performed by String 9.1 Web software, and five proteins identified in this study were marked by red arrows . Using tUBB, UBC, UBA52 and RPS27A, in the human genome. Ubiquitin is used as a covalent modifier of other proteins to activate their function and to target them for degradation, depending on the degree of ubiquitin ligation. The relevance of ubiquitination to cancer has appeared from oncogenic mutations which disrupt protein ubiquitination and control cell growth or death [Ubiquitin is a 9 kDa regulatory protein that has been found in most tissues of eukaryotic organisms. There are four ubiquitin genes, or death . Ubiquitor death . Also, tThe p53 protein is also known as cellular tumor antigen p53 (UniProt name), participates in several mechanisms of anticancer function, and plays a major role in the cellular response to a wide and diverse range of stress signals, such as DNA damage, apoptosis, genomic stability, and inhibition of angiogenesis. The p53 protein is inactivated by the negative regulator, MDM2, in the cell pathway. Upon DNA damage, metabolic or cellular stresses, polyubiquitin-26S proteasome complex will lead to the dissociation of the p53 protein and MDM2 complex and the transient p53–p21 activation will promote cell survival . Once acUbiquitin C (UBC) also participates in inactivation of mTOR, which is a master regulator of protein synthesis, translation and proliferation. The mTOR pathway may also be activated by SF-CSNP via the HER-2 receptor ,29. ActiIn particular, the PI3K/AKT pathway regulates the import and retention of glucose. It provides substrates for glycolysis and the biosynthetic pathways which rely on the supply of glycolytic intermediates. The mTOR pathway, downstream of AKT signaling, regulates the protein translation rate, and accelerates the supply of amino acid biosynthesis to generate the charged tRNAs .In our previous study of CSNPs, six proteins, Amyloid β A4 protein , C–C chemokine receptor type 8 , CD44 antigen , G2/mitotic-specific cyclin-B2 , Neuroblast differentiation-associated protein , and Osteonectin/SPARC protein , were also identified in HepG-2 cell lysate samples and associated with metastasis and cell proliferation . CD44 caet al. [g, 10 °C, 15 min) and then re-suspended in water by manual shaking. The CSNP (3 mg/mL) was added into the SF solution (3 mg/mL) slowly (12 mL/h) and stirred for 30 min (800 rpm). The SF-CSNPs were isolated by ultracentrifugation  and then re-suspended in water by manual shaking. The protein concentration of the SF-CSNPs was measured by Bio-Rad Bradford total protein assay kit .Chitosan nanoparticles (CSNP) were prepared according to Calvo et al. . BrieflyThe particle size and size distributions of the nanoparticles were performed by the particle size analyzer . For the particle size analysis, each sample was diluted to the appropriate concentration with filtered phosphate-buffered saline, pH = 7.4. Each analysis lasted 2 min and was performed at 25 °C with an angle detection of 90°.Measurement of the zeta potential of nanoparticles was performed by Zeta plus 90 particle sizer  with a 5 mW He–Ne laser (λ = 663 nm). The zeta potential values were calculated from the mean electrophoretic mobility values using the Smoluchowski’s equation.2 in RPMI 1640 medium  supplemented with 10% fetal bovine serum , 1% penicillin/streptomycin  and 44 mM NaHCO3 . After three days, the cells were washed with serum-free RPMI 1640 medium and incubated with the serum-free medium containing nanoparticles at concentrations of 1 to 5 μg/mL for 12 h.HepG2 (liver tumor cell) and CCL-13  cells were maintained at 37 °C and 5% CO5 cells/mL in 100 μL/well of appropriate cell culture media with SF-CSNPs. The cell proliferation was determined by bromodeoxyuridine assay . The cytotoxicity of SF-CSNPs was evaluated in vitro using the lactate dehydrogenase assay . These assays were performed according to the manufacturers’ instructions. The absorbance values were measured by an ELISA reader .CCL-13 and HepG2 cells were seeded in a sterile 96-well tissue culture plate at 2 × 10® cell fluorescence imaging Station, Invitrogen, Grand Island, NY, USA), and the cell micrographs were taken with a charge-coupled device (CCD) camera.For cell morphologies of HepG2 before and after incubation with SF-CSNPs, the live cell images were observed with a microscope equipped with a fluorescence light source , and cell lysates were centrifuged at 1500× Cell lysate samples (100 μL) were transferred into the 1.5 mL Eppendorf tubes and incubated at 37 °C for 3 h after mixing with 25 μL of 1 M dithiothreitol . Then cell lysate samples were reduced and alkylated in the dark at room temperature for 30 min after addition of 25 μL of 1 M iodoacetamide  in 25 mM ammonium bicarbonate. Approximately 10 μL of 0.1 μg/μL modified trypsin digestion buffer  in 25 mM ammonium bicarbonate were added to the cell lysate samples, and the cell lysate samples were incubated at 37 °C for at least 12 h in a water bath. Two μL of formic acid was added to each sample before mass spectrometric analysis for protein identification.m/z 400–2000) was followed by four data dependent tandem mass spectra with the collision energy set at 35%.The complex peptide mixtures were separated by RP-nano-UPLC–ESI-MS/MS. The protein tryptic digests were fractionated using a flow rate of 300 nL/min with a nano-UPLC system  coupled to an ion trap mass spectrometer  equipped with an electrospray ionization source. For RP-nano-UPLC–ESI-MS/MS, a sample (2 μL) of the desired peptide digest was loaded into the reverse phase column  by an autosampler. The RP separation was performed using a linear acetonitrile gradient from 99% buffer A (100% D.I. water/0.1% formic acid) to 85% buffer B (100% acetonitrile/0.1% formic acid) in 100 min using the micropump. The separation is performed on a C18 microcapillary column  using the nano separation system. As peptides eluted from the micro-capillary column, they were electrosprayed into the ESI-MS/MS with the application of a distal 2.1 kV spraying voltage with heated capillary temperature of 200 °C. Each cycle of one full scan mass spectrum (p < 0.05). Proteins were annotated by similar searches using UniProtKB/Swiss-Prot databases . The protein-protein interaction pathways were performed by String 9.1 Web software .For protein identification, Mascot software  was used to search the Swiss-Prot human protein sequence database. Positive protein identifications were defined when the Mowse scores greater than 100 were considered significant . All statistical significances were evaluated at 95% of confidence level or better. Data are presented as mean ± standard error.Previous studies have contradicting results showing whether nanoparticles are biologically compatible. In this work, a biopolymer of chitosan with silk fibroin (SF-CSNP) is introduced. CSNP was modified with silk fibroin to achieve better cell responses, and uptake by HepG2 cells was observed. Both BrdU and LDH assays showed that SF-CSNP is gentle to HepG2 cells. The same effect recurred with SF-CSNP even when the amplitude is increased. This is of interest because nanoparticles have been proposed to carry cancer drugs.To evaluate the responses of cellular proteins induced by SF-CSNP, mass spectrometry-based proteomics is adopted to analyze complex proteins of the cell lysate and to profile proteins based on their associated cell-nanoparticle interactions. By utilizing proteomic approaches, nine proteins related to SF-CSNP were identified.Although the ubiquitin proteasome/p53 pathways of the interactions between five proteins and SF-CSNPs were very unclear, the effects of SF-CSNPs to liver cells were sketched. In summary, SF-CSNPs may be nontoxic to HepG2 cells. Moreover, the functional groups of biomaterials may appear to be less damaging to the cells. This study proposed a new approach for the detection of proteins to assess the response of hepatic cells to a biopolymer. Knowing the response of cellular proteins induced by biomaterials may assist the development of nanoparticles. Although there was no direct evidence to prove the relevance between SF-CSNP and cell growth/mitosis pathways in our study, it is still worthwhile to be considered, especially as an anticancer drug delivery system.


Malignant adenomyoepithelioma and adenoid cystic carcinoma are both rare malignant epithelial-myoepithelial tumors of the breast. We report a very rare case with a malignant adenomyoepithelioma combining with adenoid cystic carcinoma in a single mass.A 61-year-old female presented with a palpable painless mass in the right breast. Mammography revealed a large irregular dense shadow without obvious internal calcification. A simple lumpectomy was performed, and a 1.6cm well-circumscribed pale-tan nodule was presented. Histologically, the nodule was composed of two obscure lobules. One exhibited typical histological image of adenoid cystic carcinoma, the other one showed the image of epithelial-myoepithelial carcinoma of salivary gland, and support the diagnosis of biphasic malignant adenomyoepithelioma. Transition between the two lobules was gradual. Immunohistochemically, CK18 and P63 highlighted the epithelial and myoepithelial cells respectively in both lesions. CD117 was positive in the epithelial cells of adenoid cystic carcinoma, but was totally negative in malignant adenomyoepithelioma.This report is, to our knowledge, the first case that combines these two tumors in a single mass. In addition, we present a review of the literature. The histogenesis of these tumors is also discussed.http://med.motic.com/MoticGallery/Slide?id=D562817E-23C2-4F72-9823-86EF6DA40005&user=2C69F0D6-A478-4A2B-ABF0-BB36763E8025 and http://med.motic.com/MoticGallery/Slide?id=38BB7126-6FFB-4B66-A208-B8C0F528DCA8&user=2C69F0D6-A478-4A2B-ABF0-BB36763E8025The virtual slide(s) for this article can be found here:  Malignant adenomyoepithelioma (AME) and adenoid cystic carcinoma (ACC) are both rare malignant epithelial-myoepithelial lesions of the breast. There are no more than 50 documented cases for the former , and theA 61-year-old female was admitted to The First Affiliated Hospital of Kunming Medical University, Yunnan, China, complaining of a lump in the right breast which had appeared 2 days earlier and had been increasing in tenderness for a week. During the physical examination, the patient’s breasts were found to be bilaterally symmetrical, without any skin retraction. The two nipples were on the same horizontal line without discharge nor retraction. A mass was palpable in the upper-outer quadrant about 5 cm away from the right nipple, with an approximate size of 1.5 cm × 1 cm. The mass was tough in texture, irregular in shape, unclear in boundary and slightly adhesive to the surrounding tissues. There were no positive findings in the left breast. The superficial lymph nodes were not palpable in the bilateral axillary and clavicular fossa. The patient had no history of smoking or alcohol consumption and there was no family history of any types of tumor.Ultrasonography revealed an ill-defined mass in the right breast, and its internal echo was non-homogenous. Mammography revealed a small irregular dense shadow in the upper outer quadrant of the right breast Figure . These hA modified radical mastectomy was performed according to the pathology results, the patient’s economic situation and with the patient’s consent. No lymph nodes involvement was found (0/21). The margins, basement membrane and nipple were all free of metastasis. Chemotherapy and radiation therapy were not indicated and the patient remained in a good condition throughout the 12-month follow-up period.th edition of WHO Classification of Tumours of the Breast [Adenomyoepithelioma is usually a benign neoplasm with a low metastatic potential. However, the malignant transformation can arise from the epithelial component, the myoepithelial component, or both. There are no well-received criteria for malignant adenomyoepithelioma because of the rarity of this lesion. According to the growth pattern of malignant components, the cases reported in the literatures can be divided into two types: 1) an area of obvious malignant component arising in a typical low-grade adenomyoepithelioma, which may be more appropriately named “adenomyoepithelioma with carcinoma” as defined in the 4e Breast , 6. Thise Breast , invasive Breast , and duce Breast , 9 whiche Breast –17  –25. In aIn this paper, we report the case of a rare biphasic malignant adenomyoepithelioma combining with adenoid cystic carcinoma in a single mass of a 61-year-old female. The histological characters of the two tumors were typical, and the transition from malignant AME to ACC was gradual. Immunohistochemistry was helpful in distinguishing these two types of malignant epithelial-myoepithelial tumors. Although most ACC has a more favorable clinical outcome, the component of high grade epithelial-myoepithelial carcinoma in this case might indicate a high frequency of recurrence and metastases.Written informed consent was obtained from the patient and her family for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


N-glycosylation is an exclusive characteristic of plant allergens not present in mammals and it could be implied in allergenic sensitization. With this aim, we evaluated and compared the allergenic activity of the protein fraction and the N-glycan fraction of the thaumatin-like protein and the main kiwi allergen, Act d 2. The natural allergen, Act d 2, was deglycosylated by trifluoromethanesulfonic acid treatment; the N-glycan fraction was obtained by extended treatment with proteinase K. N-glycan- and protein- fractions were recognized by specific IgE of kiwi-allergic patients. By contrast, the sugar moiety showed a reduced capacity to activate basophils and T cells, but not dendritic cells derived from patients' monocytes. Related to this, the production of cytokines such as IL6 and IL10 was increased by the incubation of dendritic cells with sugar moiety. Thus, the sugar moiety plays a significant role in sensitization, inducing the activation of antigen-presenting cells, but it is the protein fraction that is responsible for the allergic reactions.The physical, biochemical, and immunological characteristics of plant allergens have been widely studied, but no definite conclusion has been reached about what actually makes a protein an allergen. In this sense,  The prevalence of food allergies has increased in Western countries over the last few years. These are sometimes life-threatening and compromise the growth and development of children. In this population, many foods have to be avoided to prevent adverse reactions. The problem has come to be recognized as being more serious in recent decades because allergic sensitization, which is now known to be a strong predisposing factor for asthma, has become considerably more prevalent -fucose and/or a β-xylose residues. This N-complex glycan, highly enriched in mannose, has been associated with Th2 responses  was measured, as well as the upregulation of maturation and activation markers. The ability of the N-glycan fraction to induce T-cell proliferation was also determined.Members of the thaumatin-like protein (TLP) family are important plant allergens in several fruits, such as peach, apple, cherry, kiwi, olive, and banana, and in pollens such as cypress  with allergy to kiwi fruit Table . All patmL Eaaci .Written informed consent was obtained from all patients, and the ethics committees of the corresponding hospitals approved the study.Actinidia deliciosa fruits using the method published by our group  and using THP1-XBlue cells . These cells are derived from the human monocytic THP-1 cell line and are transfected with a reporter plasmid expressing a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-κB and AP-1.For cell cultures, the absence of LPS in the samples was checked using anti-LPS antibodies  and the reaction was carried out in an ethanol/dry ice bath, followed by incubation at −20°C for 4 h. TMSF was then removed with pyridine solution in a dry ice/ethanol bath for 15 min. The deglycosylated protein (dAct d 2) was neutralized by adding 0.5% ammonium bicarbonate and dialyzed  against H2O and then freeze-dried. The deglycosylated protein was quantified using the bicinchoninic acid test. Mus a 4 , with minor modifications. The protein was solubilized in 50 Circular dichroism measurements were performed on a Jasco Model J-715 Spectropolarimeter  using a 0.1-cm path length cell equilibrated at 20°C. Spectra were recorded for a wavelength range of 190–260 nm with 1-nm resolution at 50 nm/min scan speed and the average of three readings was calculated. The final spectra were baseline-corrected by subtracting the corresponding solvent spectra obtained under identical conditions. Results were expressed as the mean residue ellipticity (θ) for a given wavelength. The data were fitted using CDNN CD spectra deconvolution software .μg) were incubated in 0.3 mL of 0.5% (w/v) CM-Pachyman in 50 mmol/L sodium acetate, 100 mmol/L NaCl containing 0.05% (w/v) sodium azide (pH 4.0 and 5.0). A quantity of 4 mL of 0.1% (w/v) tetrazolium blue, 50 mmol/L NaOH and 0.5 mol/L sodium potassium tartrate were added to 50 μL of samples and then heated in boiling water for 3 min. The quantity of liberated d-glucose equivalents was measured as the absorbance at 660 nm. Assays were performed in duplicate. Activity was expressed as mkat/mg of protein . The reaction curve was plotted to calculate the V values. Km and Vmax were determined from a standard Lineweaver–Burke plot.For this assay, the method described by Menu-Bouaouiche et al.  was follPlectosphaerella cucumerina (106 spores/mL) were grown in 100 μL of potato dextrose broth  in 96-well microplates for 48 h at 23°C. A quantity of 20 μg/150 μL of Act d 2 or dAct d 2 proteins was added to the culture medium. The medium with fungal suspension without added proteins was used as negative control. Antifungal activity was monitored as optical densities (OD) at 660 nm every 24 h.Antifungal activity was estimated as previously described. Spores of μg/mL of proteinase K  for 24 h at 55°C and then concentrated with Amicon Ultra 3K . The unretained volume containing the sugar moiety was dialyzed against H2O . The monomer composition of the N-glycan was confirmed by strong acid hydrolysis and mass spectrometry. The total glycan was identified, and the sugar content was quantified according to Dubois' method. The N-glycan fraction was measured by matrix-assisted laser desorption/ionization (MALDI-FTMS) mass spectrometry using standard methods in the Mass-Spectrometry Services from Complutense University .Act d 2 was incubated with 5 μg of Act d 2 and dAct d 2) were separated by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and replica gels were electrotransferred onto polyvinylidene difluoride (PVDF) membranes. After blocking , membranes were incubated with a pool of patient sera (1:2 dilution in PBS) overnight, washed three times with PBS-Tween 20 0.05% for 10 min, then incubated with goat antihuman IgE-peroxidase conjugate . IgE-binding components were detected by means of chemiluminescence . Alternatively, blocked membranes were immunodetected with rabbit polyclonal antibodies to peach TLP , with anti-complex asparagine-linked glycan serum , purified Act d 2, deglycosylated Act d 2* or N-glycan fraction (5 μg/mL). For assaying carbohydrate moiety, Corning's Carbo-BINDTM plates were used following the manufacturer's instructions . Blocking solution  without solid phase was used as a negative control. Bromelain was used as an allergenic sugar control. Those assays yielding ODs > 0.12 units  were considered positive. All tests were performed in triplicate.ELISA assays were carried out as previously described , we added 50 μL of stimulation buffer , containing IL-3 (2 ng/mL) and heparin  in the cell suspension. As a positive control, a monoclonal anti-IgE antibody was used at a final concentration of 1 μg/mL. A positive response was considered for stimulation index  ≥2.This test was performed as previously described  for proliferation analysis in Roswell Park Memorial Institute (RPMI) medium , supplemented with 10% (v/v) of fetal calf serum (Invitrogen), 0.02 mmol/L mercaptoethanol, 2 mmol/L of glutamine, and 10 mmol/L HEPES. To evaluate cell proliferation, PBMCs were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) . The results were considered positive when the SI—calculated as the ratio between %CFSElow (with stimulus)/%CFSElow (without stimulus)—was >2. Data presented are the values obtained corresponding to antigen concentration of 5 μg/mL.Peripheral blood mononuclear cells were purified from six kiwi-allergic patients, as previously described  with l-glutamine, 10% heat-inactivated fetal bovine serum , and 100 mg/mL antibiotics  following the manufacturer's instructions, and with 200 ng/mL rhIL4 and 100 ng/mL rhGM-CSF , for 4–5 days at 5% CO2 and 37°C, as described previously  with Act d 2, dAct d 2, and N-gly at 20 μg/mL. LPS at 10 μg/mL (Sigma) was used as a positive control. After 72 h of stimulation at 37°C in 5% CO2, treated and nonuntreated DCs were recovered and maturation was assessed by CD83 , CD80 and CD86 upregulation (Immunotools), in an Accuri cytometer . The maturation index (MI) was calculated as the ratio between stimulated and unstimulated DCs. A response was considered positive when MI > 2.Monocytes were purified from PBMCs of six kiwi-allergic patients and six healthy donors by positive selection using CD14 dynabeads , following the manufacturer's protocol. DCs were derived from monocytes by culturing the CD14α, and IL-1β of moDCs was determined by real-time PCR, after incubating the cells with 20 μg Act d 2, dAct d 2, and N-gly for 72 h. Cells were recovered and mRNA was isolated according to the Qiagen- RNeasy protocol  and stored at −80°C. RT-PCR was performed as previously described  and relative quantification was performed using the comparative threshold cycle method (2Ct−DD), as described by Livak and Schmittgen  was considered to be significant in all analyses.Data were expressed as means ± SDs of two or more independent experiments. Statistical analysis was performed using SPSS 17.0  and Statgraphics Centurion XVI . Comparison between natural Act d 2 and protein/sugar fractions was carried out using Wilcoxon paired samples test or Mann–Whitney N-glycan complex was assembled and refined with Chimera  by means of TMSF , antibodies against xylose (anti-xyl) or by antibodies against fucose (anti-fuc), suggesting that deglycosylation treatment successfully removed the glycan fractions .In the same way, the far-ultraviolet CD (far-UV CD) of deglycosylated protein, dAct d 2, showed an overlapping spectrum with no differences compared to the natural protein, Act d 2 . The natural and deglycosylated forms had overlapping kinetic curves . The deglycosylated form preserved the ability to inhibit the growth of P. cucumerina, although this inhibitory capacity was lower than in the case of the natural protein .The enzymatic activity of the deglycosylated protein was measured by the ate Fig. C. dAct dN-glycan to IgE recognition of Act d 2, this fraction was isolated by extensive treatment of the natural allergen with proteinase K. The purity of the sugar fraction was confirmed by thin layer chromatography (data not shown) and mass spectrometry  were used as a solid phase to determine the specific IgE levels of single sera from 24 kiwi-allergic patients. Positive response to naïve allergen (Act d 2) was observed in all patients, while 16 of them (66.7%) recognized both fractions separately . By contrast, nts Fig. B.N-glycan (N-gly)  or together with one of the isolated fractions, that is, the protein (dAct d 2*) or the ly) Fig. . The proN-glycan fraction (N-gly) and the natural allergen (Act d 2) .Monocyte-derived dendritic cells from six healthy donors and six allergic subjects were grown in the presence of increasing concentrations of deglycosylated protein (dAct d 2),  2) Fig. A. When tU test, P = 0.2080). By contrast, the protein fraction dAct d 2 showed a reduced capacity to induce maturation of moDCs, showing statistical differences with respect to the natural allergen  and N-glycan fraction .Both sugar and naïve allergen induced the expression of the maturation markers CD80, CD83 and CD86, with no significant differences observed among them  have been studied, as have their heat and protease resistance properties. However, the day we can predict whether a protein is allergenic remains a long way off.N-glycosylation in allergenic activity, taking Act d 2  as a model. We isolated the protein and N-glycan fractions of this allergen. The deglycosylation treatment of Act d 2 did not alter its enzymatic activity or its antifungal capacity, and the far-UV CD indicated that its secondary structure was maintained.In order to understand the sensitization mechanism, in this study we evaluated the contribution of N-glycan fraction of Act d 2 revealed a mixture of sugars. Mass spectrometry showed at least seven peaks between 1100 and 3000 Da. Searches of specialized sugar databases suggested several possible structures. The heterogeneity of these types of sugar could interfered in the match of TLPs as unique bands by SDS-PAGE, which may often be misinterpreted as a lack of purity in the samples  -mannose on M3X(F) structures might physically interfere with the binding. When N-glycan structures of the three major aeroallergens, Ole e 1, Ara h 1, and Lol p 11 were analyzed it was found that each one corresponded to at least two isoforms of sugar. The most extreme case was that of Ara h 1, which was found to be associated with at least four types of glycosylation, varying in their mannose and xylose content -fucose and β--xylose in the IgE-binding capacity of the complex plant N-glycans. In our case, the monomer composition of the N-glycan fraction was confirmed by strong acid hydrolysis and mass spectrometry. This fraction contained monomers of mannose, N-acetyl glucosamine, xylose, and fucose, suggesting the presence of typical N-plant glycosylation.Generally, plant glycoproteins prepared from biological sources are characterized by a mixture of trimmed and untrimmed N-glycan fractions revealed that most patients recognized both fractions. More than 77% of kiwi-allergic patients presented specific antibodies against both fractions, as shown by ELISA assays. By contrast, this recognition was not based on N-glycosylation cross-reactivity, because the bromelain (Ana c 2), commonly used as a glycoprotein control, was recognized by fewer than 17% of patients. In other words, N-glycan fraction was specifically IgE recognized.The analysis of the IgE-binding capacity of the protein and N-glycan fraction was shown not to play an important role in the basophil activation of patients to kiwi. This suggests that many patients may be sensitized to N-complex sugars of plants, but suffer no symptoms when they are exposed to them. CCDs are clinically irrelevant for most of the patients. According to the data, in a minor, but significant fraction of patients (10%) CCDs sensitivity might play a role in clinical allergy.However, this specific IgE to N-glycan fraction could induce maturation of antigen-presenting cells, such as moDCs, but could not activate PBMC proliferation. In fact, the culture of N-glycan fraction with moDCs induced the production of cytokines such as IL6 and IL10. These results suggest that the sugar may be able to stimulate an immune response from antigen-presenting cells, but the protein fractions  could only induce a T-cell response.Conversely, we observed that the N-glycan fraction can be recognized by dendritic cells is well understood (Altmann N-glycan structure that was rich in fucose and xylose. It is possible that the mechanism involved in recognizing the N-glycan fraction of Act d 2 is comparable to that of Ara h 1.The mechanism by which the  Altmann . Many of Altmann , whereas Altmann . The dif Altmann  comparedN-glycan fraction in the allergic sensitization to Act d 2. N-glycan does not appear to contribute to T-cell activation or the allergic cellular response. In summary, N-complex plant glycosylation may play an essential role in the activation of antigen-presenting cells and appears to trigger a proinflammatory response contributing toward the development of a Th2 response. By contrast, once the allergy is established, the N-glycan involvement in the induction of symptoms as well as in T-cell activation would be negligible. These findings provide useful information that is likely to influence current allergy immunotherapy strategies.In summary, this study shows the importance of the


Stachybotrys elegans is able to parasitize the fungal plant pathogen Rhizoctonia solani AG-3 following a complex and intimate interaction, which, among others, includes the production of cell wall-degrading enzymes, intracellular colonization, and expression of pathogenic process encoding genes. However, information on the metabolome level is non-existent during mycoparasitism. Here, we performed a direct-infusion mass spectrometry (DIMS) metabolomics analysis using an LTQ Orbitrap analyzer in order to detect changes in the profiles of induced secondary metabolites of both partners during this mycoparasitic interaction 4 and 5 days following its establishment. The diketopiperazine(s) (DKPs) cyclo(S-Pro-S-Leu)/cyclo(S-Pro-S-Ile), ethyl 2-phenylacetate, and 3-nitro-4-hydroxybenzoic acid were detected as the primary response of Rhizoctonia 4 days following dual-culturing with Stachybotrys, whereas only the latter metabolite was up-regulated 1 day later. On the other hand, trichothecenes and atranones were mycoparasite-derived metabolites identified during mycoparasitism 4 and 5 days following dual-culturing. All the above secondary metabolites are known to exhibit bioactivity, including fungitoxicity, and represent key elements that determine the outcome of the interaction being studied. Results could be further exploited in programs for the evaluation of the bioactivity of these metabolites per se or their chemical analogs, and/or genetic engineering programs to obtain more efficient mycoparasite strains with improved efficacy and toxicological profiles. This involves a sequence of changes in the metabolism of both partners. Focusing on crop protection, mycoparasitism holds the premise of becoming a valuable component of integrated pest management strategies (IPM)  analysis using a linear trap quadrupole (LTQ) Orbitrap Classic analyzer. Moreover, because metabolite identification represents a bottleneck for fungal metabolomics, , and water (HPLC grade) were purchased from Fisher Scientific Company .Stachybotrys elegans (Pidoplichko) W. Gams (ATCC 18825) and the pathogen Rhizoctonia solani AG-3 (ATCC 10183) were revived from pre-colonized oat kernels on 1% potato dextrose agar  and incubated at 24°C for 7 and 5 days, respectively. Induction and collection of S. elegans conidia were performed as previously described  composed (g L−1) of: MgSO4.7H2O, 0.2; K2HPO4, 0.9; KCl, 0.2; FeSO4.7H2O, 0.002; MnSO4, 0.002; ZnSO4, 0.002; NaNO3, 1.0; biotin, 10 mg; gellan gum, 1%  .Dual-culturing of R. solani culture were grown on MSMA for 48 h and then sprayed with 100 μL of a suspension of S. elegans conidia (106 mL−1 water) using a Badger 350 air brush and MC-80 mini air compressor calibrated at 1 kg cm−2. The control treatments consisted of spraying 100 μL of S. elegans conidia on non-inoculated MSMA plates and R. solani-inoculated MSMA plates sprayed with sterile distilled water. Additionally, a negative control representing the MSMA medium was used to determine compounds of non-biological origin. All culture plates were incubated at 24°C for 4 or 5 days following dual and pure strain cultivation. These time points were chosen based on a priori knowledge to capture the infection and colonization of R. solani hyphal cells by S. elegans  of a 5-day old To associate the metabolic changes with the progress of the mycoparasitic process, agar pieces (5 × 5 mm) from interaction zones of dual-culture plates and from pure cultures of both fungal partners were collected in a time course. Sections from interacting zones were stained with lactophenol blue or water and viewed under an optical microscope. Presence of hyphal coils, penetration pegs and intracellular colonization of the pathogen was digitally documented with the Moticam 2300 digital camera .2, and samples were stored at -80°C until further processing. Extraction was performed as previously described  and negative (ESI−) electrospray modes. Finally, extracts were dried using a Labconco CentriVap refrigerated vacuum concentrator equipped with a cold trap .Four plugs (8 mm in diameter × 7 mm in height) were collected from the interaction zones of dual-cultures, pure cultures of each fungal partner after 4 or 5 days of cultivation and from the negative control (MSMA) plates. Plugs were placed in glass autosampler screw thread vials . Quenching was instantly performed by adding twice 2 mL of liquid N+ or ESI− modes   or methanol/ammonium acetate (4 mM) was added to the dried samples, respectively. Extracts were then transferred to glass microinserters (150 μL), which were consecutively placed into 2 mL glass autosampler vials. Samples (10 μL) were injected at a flow rate of 10 μL min−1 using a 100 μL syringe . Full scan mass spectra were acquired in the range between 50 and 1200 Da at a rate of 0.6 scans/s and a mass resolution of 60,000 at 400 m/z. The source and capillary voltages were set to 3.2 kV and 5.0 V for ESI+ and 4.0 kV and −35 V for ESI−, respectively. The capillary temperature for both modes was set to 275°C. Sheath gas flow was set to 10 (ESI+), and 20 (ESI−) whereas no auxiliary and sweep gases were used. For selected samples, MS/MS spectra were recorded using previously described settings  was used acquiring in the ESI+ and 0.6–1.1 min for ESI−. Metabolic features were detected using the centroid algorithm and the noise level was optimized for each sample. The Fourier transform mass spectrometer (FTMS) shoulder filter was then applied at a mass resolution of 8000 using the Lorentzian extended model function. Chromatogram built, alignment and gap-filling were performed using an m/z tolerance (Δppm) <3. Alignment was performed using the “Join aligner” option, whereas gap filling was performed in two steps; first using the “Peak finder option” and then the “Same RT and mz range gap filter”  for multivariate analysis . Based on the variability in the model parameters encountered in the different cross-validation cycles, standard errors were calculated with 95% confidence interval using Jack-knifing  and PubChem (http://pubchem.ncbi.nlm.nih.gov/). Identification of metabolites was based on mass accuracy (<2 ppm) and where available, on isotope and/or MS/MS fragmentation patterns (Supplementary Data Sets http://metlin.scripps.edu/index.php) and mzCloud (https://www.mzcloud.org/) and the literature. In addition, the heuristic rules of Kind and Fiehn  . In addition, the possible presence of metabolites with identical molecular formulae or isomers makes their absolute identification even more complex. In this context, the identification of metabolites during the mycoparasitic interaction being studied, was based on searches against the two species-specific metabolite libraries for  ppm. In R. solani-S. elegans dual-cultures were compared to those of pure cultures for both time points   or elimination following filtering . The latter could be attributed either to the variation in the biological samples or signals with intensity near the limits of detection of the instrument.Stachybotrys mycoparasitic activity on Rhizoctonia's metabolism  cyclo(S-Pro-S-Leu)/cyclo(S-Pro-S-Ile), ethyl 2-phenylacetate, and 3-nitro-4-hydroxybenzoic acid /cyclo(S-Pro-S-Ile) do not exhibit phytotoxicity  and its derivatives are the first studied bioactive metabolites of Stachybotrys to overcome the defense mechanisms of Rhizoctonia and parasitize it is indirectly linked to the suppression of the biosynthesis of the majority of its metabolites with well-established bioactivity. Among these, melatonin is an antioxidant and free radical scavenger in many organisms, including fungi , with the exception of the macrocyclicroridin A-trichoverrin A/B (Supplementary Data Set Stachybortys-produced atranones D/E, F and H, were identified during mycoparasitism, with the latter being present only during mycoparasitism and not in pure cultures (Supplementary Data Set de novo or substantially increased biosynthesis during mycoparasitism. This toxin is an analog of atranones A, B, and I (Hinkley et al., Stachybotrys spp. and Myrothecium verrucaria (Bräse et al., In addition to trichothecenes, the Stachybotrys metabolites were detected only in pure cultures (Supplementary Figure Rhizoctonia-derived metabolites, and needs further investigation.Finally, a small number of identified R. solani-derived metabolites were down-regulated in dual-cultures possibly due to the direct effect of the mycoparasite on host's metabolism or because they were produced in trace amounts. Alternatively, S. elegans mycotoxins known as trichothecenes were up-regulated during mycoparasitism. To the best of our knowledge, this is the first report on the involvement of trichothecenes in the active process of mycoparasitism. Results could be further exploited in programs for the evaluation of the bioactivity of these metabolites per se, or their structures as chemical analogs and/or genetic engineering programs to obtain more efficient mycoparasite strains with improved efficacy and toxicological profiles. Experiments are underway to isolate the most induced metabolites from each fungal partner and test their bioactivity against each other.An original DIMS metabolomics approach was developed for the monitoring of the production of secondary bioactive metabolites in interaction zones of hyphal mycelia formed between a mycoparasite and a fungal pathogen during active mycoparasitism. In these zones both partners are subjected to intense stress leading to the induction of secondary bioactive metabolites for attack and/or defense. The majority of the antimicrobial RC, KA, and SJ conceived, designed and executed the experiments. RC and KA analyzed the data. RC, KA, and SJ contributed to the writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


KA) on the GOS film-based SPR chip and the conventional SPR chip for 100 μg/ml BSA are 80.82 × 106 M-1 and 15.67 × 106 M-1, respectively. Therefore, the affinity constant of the GOS film-based SPR chip is 5.2 times higher than that of the conventional chip. With respect to the protein-protein interaction, the SPR sensor capability to detect angle changes at a low concentration anti-BSA of 75.75 nM on the GOS film-based SPR chip and the conventional SPR chip is 36.1867 and 26.1759 mdeg, respectively. At a high concentration, anti-BSA of 378.78 nM on the GOS film-based SPR chip and the conventional SPR chip reveals two times increases in the SPR angle shift. Above results demonstrate that the GOS film is promising for highly sensitive clinical diagnostic applications.This work develops a highly sensitive immunoassay sensor for use in graphene oxide sheet (GOS)-based surface plasmon resonance (SPR) chips. This sensing film, which is formed by chemically modifying a GOS surface, has covalent bonds that strongly interact with the bovine serum albumin (BSA), explaining why it has a higher sensitivity. This GOS film-based SPR chip has a BSA concentration detection limit that is 100 times higher than that of the conventional Au-film-based sensor. The affinity constants ( The ratio of the affinities of the ODT-based GOS film-based SPR chip, conventional chip, and GOS film SPR chip was 1:6:31 times. The results demonstrate that this Cys-modified Au surface excellently immobilized a GOS film in an SPR chip.Molecular kinetics of the interactions of the three sensor films and the protein are analyzed. Figure y = -6.43 + 2.77 ex0.54  for the GOS film-based SPR chip, and y = -1.9 + 0.12 ex0.87  for the conventional SPR chip, where x is the concentration of BSA and y is the SPR angle (θ).Figure To evaluate the sensitivity and specificity of the developed immunoassay film in the on-site detection of anti-bovine serum albumin , an anti-BSA antibody sample was diluted to 378.78, 151.51, and 75.75 nM by adding PBS buffer. Figure x-0.034 for the GOS film-based SPR chip and 92.312 to 82.146 ex-0.0035 for the conventional SPR chip.Binding affinity was determined using anti-BSA protein concentrations of 75 to 378.78 nM. Since the immunoassay analyses were carried out using the same protein, BSA, with the anti-BSA interaction, the results are similar to those of the kinetic analysis, as shown in Figure In summary, a GOS film was developed for binding with proteins based on SPR analysis for the purpose of immunoassay sensing. The GOS film-based SPR chip herein had a BSA concentration detection limit of as low as 100 pg/ml, which was 1/100th that of the conventional SPR chip. Additionally, in immunoassay detection, the GOS film-based SPR chip was highly sensitive at a low concentration of 75.75 nM, exhibiting an SPR angle shift of 1.4 times that of the conventional chip, and exhibited an SPR angle shift of two times that of the conventional chip at a high concentration of 378.78 nM. Finally, we believe that the fact that the GOS can be chemically modified to increase its SPR sensitivity can be exploited in clinical diagnostic protein-protein interaction applications, especially in cases in which tumor molecular detection is feasible.The authors declare that they have no competing interests.N-FC participated in the design of the study and performed the statistical analysis and drafted the manuscript. T-YH carried out the immunoassays and performed the statistical analysis. H-CL and K-CL conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.


AbstractAlthough acute myeloblastic leukemia (AML) is more resistant to chemotherapy than acute lymphoblastic leukemia (ALL), significant progresses have been achieved over the last 20 years with an improvement in the long-term survival up to 50-60%. This may be attributed to the intensification of chemotherapy, including the increased use of stem-cell transplantation (HSCT) in well-defined subgroups. Allo-HSCT represents an extremely effective alternative in pediatric AML treatment panel, but its efficiency is limited both by the toxic effects and by the difficulty of finding a matched HLA donor. The application of the hematopoietic stem cells transplantation (HSCT)  is indicated in CR1 only in patients in the high-risk group (HR); there are studies that demonstrate the allo-HSCT (matched sibling donor) superiority vs. chemotherapy, and other studies that prove that there is no statistically significant difference.  According to the current evidence, HSCT .The bone marrow harvesting procedure is done while under general or epidural anesthesia. The stem cells quantity which was harvested is expressed as a number of nucleate/kgc cells . The bone marrow is harvested in anticoagulant blood bags (heparin 5U/mL marrow), which must be periodically stirred in order to avoid clots formation. Afterwards a leukocyte count is done, which evaluates with a bigger precision the volume of the harvested bone marrow  and the history of acute GvHD. Although total immunoglobulins are normal or even raised, the level of IgA, IgG and subclasses IgG are decreased and the opsonization and the reticulum-endothelial function are altered. The restoration of immunity after chronic GvHD needs many years, being a process that takes place gradually.  Although rarely met in children compared to the adults, chronic GvHD – limited or extensive – develops at >100 days post-transplant, also appearing starting from 40 days post-transplant. Chronic GvHD has the aspect of an autoimmune disease of the conjunctive test , being associated with deficiencies of the cellular and humoral immunity, deficiencies of the macrophages, PMN chemotaxis disorders, altered answer to vaccinations and severe mucositis. The risk factors for chronic GvHD development are age, allogenic transplant  [The prophylaxis recommendations of the graft-versus-host-disease (GvHD) according to the AIEOP study group are the following:  Transplant from a family donor who is HLA-identical:  • Cyclosporine-A: 2 mg/Kg/day i.v., in 2 administrations, from the 3rd day until approximately in day +20 - +25 post-transplant. It is orally administered successively in doses of 4 – 6 mg/Kg/day. After 6-8 months post-transplant, the immunosuppressive treatment can be gradually decreased to 1 year post-transplant. The Cyclosporine A plasmatic level must be monitored and must be maintained between 100 and 250 ng/mL.  • Methotrexate administered on short-term: 15 mg/m2 i.v. in 30 minutes from day +1; 10 mg/mp i.v. in 30 minutes in day +3 and +6 post-transplant.In the case of unrelated voluntary donor transplant, the following are recommended: • Cyclosporine-A: administered according to the same dosage as in the case of a family donor who is HLA identical• Methotrexate administered on short-term: 15 mg/m2 i.v. in 30 minutes from day +1; 10 mg/mp i.v. in 30 minutes from day +3, +6, +11 post-transplant. • ATG: Fresenius 5 -10 mg/Kg/die i.v. or Genzyme 2 – 3 mg/Kg/day, in vep 6 – 8 hours, from the 5th day to the -3 day. Premedication which includes methylprednisolone and antihistamines.


Crassostrea gigas. With numerous analyses, it was identified and characterized that both the nacrein-like proteins F1 and F2 were secreted and most closely related to the nacrein-like protein of California mussel Mytilus californianus via phylogenetic analysis. RT-PCR analysis showed that the nacrein-like proteins F1 and F2 were expressed in multiple tissues and the expression levels remarkably rose after entering the spat stage, which were basically consistent with the increase of calcite fractions in the total shell volume. Surprisingly, the Gly-X-Asn repeat domain, which is distinctive in most nacrein-like proteins, was absent in the two newly identified nacrein-like proteins in C. gigas and replaced with a series of acidic amino acids (D/E). Regardless, nacrein-like proteins in mollusks seem to be vital to the deposition of calcium carbonate and likely perform diverse functions.Nacrein-like proteins have carbonic anhydrase (CA)-like domains, but their coding regions are flanked by inserted repeat sequence, such as Gly-X-Asn. Reportedly, nacrein-like proteins show the highest similarity to human carbonic anhydrase 1(α-CA1), possess CA catalytic functions, and play a key role in shell biomineralization. In the present study, two novel nacrein-like proteins were firstly identified from the shell-forming mantle of the Pacific oyster The online version of this article (doi:10.1007/s11033-014-3298-z) contains supplementary material, which is available to authorized users. The mollusc shell is a remarkable model of matrix-mediated mineralization performed outside the living tissues. The shell organic matrix mainly consists of proteins, chitin, and polysaccharides that can precisely self-assemble, which is considered as a key role in crystal polymorphism, nucleation, growth, and termination to form the final texture of the shell . Thus, tPinctada fucata, was found to be specifically involved in nacreous layer formation in the shell and pearl [P. fucata [Pinctada maxima) [Turbo marmoratus) [Crassostrea nippona), Yesso scallop (Patinopecten yessoensis) [Tridacna gigas) [P. maxima) [Nacrein, the first identified organic matrix component in the Japanese pearl oyster nd pearl . Nacrein. fucata . Nacrein maxima) , turban moratus) , the edisoensis) , giant ca gigas) , 9, and  maxima) , 7, 10. Crassostrea gigas  is a marine bivalve belonging to the phylum Mollusca, and has long been as an interesting model for developmental biology. Regarding shell biomineralization, C. gigas also can be considered as an experimental model for several unique characteristics. For one thing, except for five small, distinct, well-defined areas consisting of aragonite, the adult shell of C. gigas mostly consists of calcite crystals [Pinctada nacre model. For another thing, the inner shell microstructure of C. gigas is composed solely of foliated and chalky structures, while it is more diverse in the pearl oyster [The Pacific oyster crystals , 12, whil oyster . Furtherl oyster , 15.C. gigas and to describe their structures and evolutionary relationships with all known nacrein-like proteins and CAs already identified in the mollusks. Our results revealed that the diversity in shell organic matrix compositions maybe decide the variety of shell structure.The present study is the first to report the complete sequences of two novel nacrein-like proteins isolated from the shell-forming mantle of Wild adult Pacific oysters were collected from the aquatic farm in Laoshan  and healthy individuals were selected and cultivated in aerated seawater at 23 °C for several days prior to use.3 bucket  of filtered seawater and incubated at 23 °C. The larvae were observed via light microscopy, harvested at appropriate stages, and then rapidly frozen in liquid nitrogen along with one sample of unfertilized eggs. The salinity of the filtered seawater used in the experiments was ~30 ppt.Larva oysters of different developmental stages were cultured from a hatchery located in Laoshan  using an insemination technique modified from the methods described by Fujimura et al. . Eggs fr® reagent (Invitrogen) with the manufacturer’s protocol, and quantified using a NanoDrop spectrophotometer , and then stored at −80 °C for further use. The integrity of the RNA samples was also evaluated by agarose gel electrophoresis. Reverse transcription was performed with 1 μg of total mRNAs using the Prime Script RT reagent Kit with gDNA Eraser (TaKaRa) to avoid genomic DNA contamination, and dT-AP served as the reverse transcription primer.Total mRNAs were extracted from the shell-forming mantle of wild adult oyster using TrizolTwo sequences described as nacrein-like proteins after sequence homology analysis were retrieved from the oyster genome database. Firstly, two pairs of test-primers were designed based on the two sequences and used to obtain the nacrein-like protein F1 and F2 transcripts. The extracted PCR products were subcloned into a pEASY-T1 vector (TransGen), and positive clones were sequenced. Secondly, the complete 3′ and 5′ end sequences were obtained via nested-PCR using 3′ and 5′ RACE primers, which were designed according to the above nucleotide fragments. In the 3′ RACE nested-PCR, AP/3RACE-F1 and AP/3RACE-F2 were used to obtain a complete 3′ end sequence, including the polyadenylation (poly (A)) signal. The cDNA templates used in the 5′ RACE nested-PCR were required to be purified and add poly (C) to the 5′ end in advance, then primers dg-AP/5RACE-R1 and dg-AP/5RACE-R2 were employed to obtain a complete 5′ end sequence. The PCR products were subcloned and sequenced by the method described above. Finally, the full length cDNA sequences were confirmed using gene-specific primers, which were designed based on the untranslated regions of the 5′ and 3′ terminals, respectively. The synthesis of all PCR primers and cDNA sequencing were completed by Sangon Biotech (Shanghai) and all the primer sequences are listed in Table S1.C. gigas were identified as being nacrein sequences using tblastx analysis in NCBI database (http://www.blast.ncbi.nlm.nih.gov/) and then analysed in silico using various tools from the Centre for Biological Sequence Analysis database (http://www.cbs.dtu.dk/services/). The deduced amino acid sequences were compared together with six nacrein-like proteins reported in mollusks and CA1 of Homo sapiens using the ClustalX .The two full-length cDNA sequences in Sequences of 18 nacrein-like proteins from bivalves, one nacrein-like protein from gastropod, and 16 CAs from mollusks were aligned using the ClustalX. The alignment was manually modified using Bioedit software and Gly-X-Asn repeats in all nacrein sequences were removed for the alignment. The Phylogenetic Estimation using Maximum Likelihood algorithm with a BIONJ-defined starting tree .C. gigas  and seven stages of C. gigas . Total mRNAs (1 μg) were extracted and quantified as described above. For each gene, RT-primers were designed (Table S1) and tested using serially diluted total mRNAs  according to the original concentration. The gel pictures were analysed to verify the specificity of the amplified products. Real-time PCR was performed using an ABI 7500 Fast instrument and 7500 software version 2.0.1 . Each sample from the three specimens was amplified in triplicate and a negative control was analysed in parallel for each gene. Elongation factor 1 alpha  and ribosomal protein S18  were used as reference genes in the adult and larva samples, respectively [−∆∆CT method [Real-time PCR analyses were both performed on cDNAs from seven tissues of three adults T method .The nacrein-like protein F1 consists of 1,631 bp encoding 428 amino acids from a 1284-nucleotide open reading frame, ranging from the ATG translation initiation codon to the TAA stop codon. The nacrein-like protein F2 contains a 1287-nucleotide open reading frame encoding 429 amino acids, with the translation initiation codon ATG and the stop codon TGA. The two nucleotide sequences were deposited in the GenBank database under the accession numbers KC563208 and KC563207, respectively.Blastp analyses clearly showed that the two sequences were members of the α-CA family and each possessed a conserved CA catalytic domain inserted by a repeat sequence of acidic amino acids (D/E) . In particular, three histidine residues essential for the zinc cofactor binding and 25 active residues possessing a critical function in CA activity both appeared in the two sequences , indicating that they had an active function in the shell-forming mantle . The priC. gigas showed the highest similarity with the nacrein-like protein in Mytilus californianus, followed by nacrein in P. fucata, although the similarity is comparatively low in the Gly-X-Asn repeat domain. What’s more, the two nacrein-like proteins of C. gigas share several conserved peptides that are typical among all nacrein-like proteins, such as QSPIN, GSEHS, PMEA, YTYE/A/PGSLT/STPPC, and three histidine residues involved in zinc binding .A search of the UniProtKB/Swiss-Prot database revealed that the both two new sequences of Mytilus californianus. The scale bar indicates 1.1 substitutions per amino acid site.A phylogenetic tree was created from the alignment of 35 nacrein-like proteins and CAs among mollusks using the maximum likelihood method Fig. . OverallC. gigas, although the maximum relative expression levels between the right and left mantles were unequal. The transcript level of nacrein-like protein F2 was also abundant in the hematocyte, but lower expression was detected in the gill and gonad, which was dissimilar to that of a true CA activity, which was the highest in the gill [Real-time PCR analyses of different tissues Fig. a showed the gill . The expC. gigas, which were named nacrein-like proteins F1 and F2, respectively. As other nacrein-like proteins in bivalves, the two both exhibit typical catalytic sites of α-CAs with three histidine residues and 25 active cites [In silico analyses of the primary structure revealed the presence of signal peptides and N-terminal transmembrane regions both in F1 and F2, indicating that the two are secreted CAs. It was reported that phosphoryl and saccharide chains might have a close relationship with the biomineralization process, as the phosphorylation of amino acids could change the tertiary structure of the protein and subsequently its interactions with Ca2+ [2+. The saccharide chains of F1 and F2 probably have the similar effects.The present report is the first to identify two new shell matrix proteins from the shell-forming mantle of the Pacific oyster ve cites   both in the CA and Gly-X-Asn -repeat domains. The component isolated from P. fucata can also be classified into this type [Nacrein contains a CA domain as well as a Gly-X-Asn repeat domain, and the latter is supposed to interact with calcium carbonate, as indicated by its inhibited precipitation in an in vitro crystallization experiment . Howeverhis type .As shown in Fig. C. gigas lives attached to solid objects through the left shell, thus the differential expression between the left and right mantle may relate to its attachment behavior. We also investigated the expression levels of nacrein-like protein F1 and F2 transcripts during the ontogeny of C. gigas Fig. S1Homo sapiens were edited using Bioedit software. Conserved amino acids have black (100%) or dark gray (70%) backgrounds, whereas nonconserved amino acids have a white background. The putative active sites of the functional domains in the CA1 (Homo sapiens) are indicated by diamonds (◊), among which 25 active sites appear in the nacrein-like proteins of C.gigas, including three histidine residues that bind to zinc ions(◊). Signal peptides and the transmembrane region in nacrein-like proteins F1 and F2 are boxed and highlighted in yellow, respectively. Amino acids in pink background indicate putative phosphorylation sites ; Characters highlighted in green indicate putative N-glycosylation sites . Accession numbers: Homo sapiens: CA1/ AAH27890; Crassostrea gigas: nacrein-like protein F1, F2 and F3; Pinctada maxima: nacrein like protein (pm) /BAF42330.1; Mizuhopecten yessoensis: nacrein-like protein P1/BAF42331.1; Pinctada fucata: nacrein/BAA11940.1; Pinctada margaritifera: nacrein-like protein C5/AEC03973; Mytilus californianus: nacrein-like protein/P86856.1 and Turbo marmoratus: nacrein/ BAB91157 (PDF 132 kb)Alignments of nine nacrein-like proteins from seven mollusc species and CA1 from Below is the link to the electronic supplementary material.


In patients with community-acquired pneumonia (CAP), short-term mortality is largely dependent on pneumonia severity, whereas long-term mortality is considered to depend on comorbidity. However, evidence indicates that severity scores used to assist management decisions at disease onset may also be associated with long-term mortality. Therefore, the objective of the study was to investigate the performance of the pneumonia severity scores CURB-65 and CRB-65 compared to the Charlson Comorbidity Index (CCI) for predicting 1-year mortality in adults discharged from hospital after inpatient treatment for CAP.From a single centre, all cases of patients with CAP treated consecutively as inpatients between 2005 and 2009 and surviving at least 30 days after admission were analysed. The patients’ vital status was obtained from the relevant local register office. CURB-65, CRB-65 and CCI were compared using receiver operating characteristics (ROC) analysis.Of 498 cases analysed, 106 (21.3%) patients died within 1 year. In univariate analysis, age ≥65 years, nursing home residency, hemiplegia, dementia and congestive heart failure were significantly associated with mortality. CURB-65, CRB-65 and CCI were also all associated with mortality at 1 year. ROC analysis yielded a weak, yet comparable test performance for CURB-65  and CCI .Neither CURB-65 or CRB-65 nor CCI allow excellent discrimination in terms of predicting longer term mortality. However, CURB-65 is significantly associated with long-term mortality and performed equally to the CCI in this respect. This fact may help to identify CAP survivors at higher risk after discharge from hospital. Community acquired pneumonia (CAP) is the major cause of mortality from infectious diseases in western countries. In 2011, around 20,000 patients in Germany died due to pneumonia and influenza ,4. SeverBesides remarkable hospital mortality, research from the recent years shows alarming long-term mortality among patients who were discharged as clinically recovered after a CAP episode -21. LongApart from comorbidity, several studies report that pneumonia severity on admission has also an influence on long-term mortality ,16,17,25We conducted a retrospective study at the Marienhospital Herne, Herne, Germany. Some study details and results on short-term mortality have already been published . Data ofTo validate CAP diagnosis, all cases in the database were reviewed. We included those cases with typical clinical symptoms of pneumonia  and with either newly shown pulmonary infiltrate on chest x-ray or elevated biochemical markers of inflammation within 48 hours of hospital admission with no evidence of other infectious diseases. Patients who had undergone radiotherapy or chemotherapy within 28 days prior to admission or with known immunodeficiency of other causes were excluded. Patients with nosocomial pneumonia and pneumonia due to other causes, e.g. stenosis-induced pneumonia or pneumonia following pulmonary embolism, as well as patients suffering from acute exacerbation of chronic obstructive disease were also excluded. Patients who had not undergone or discontinued treatment, those referred from other hospitals and patients on mechanical ventilation upon admission were considered ineligible for inclusion. The study was restricted to inpatients who survived the initial admission for CAP for at least 30 days.For the purpose of this study, we used a dichotomous risk factor for 1-year mortality, ‘low risk’ versus ‘high risk’, as defined, for example, by CCI or CURB-65. On the basis of data from Hsu et al. , we assuCategorical variables are presented in absolute numbers and proportions, continuous variables with median, mean and range. Risk stratification for all patients was calculated on the basis of CURB-65, CRB-65 and CCI by score points and risk categories, respectively. The association between categorical variables was assessed using a chi-square test. For multivariate analysis, logistic regression analysis was used, with one model for each of the scores in use. In all models, death within 365 days after hospital admission was the dependent variable, coding ‘1’ for death and ‘0’ for being alive. As explanatory variables, we used age , sex and the score in three categories, respectively. Receiver operating characteristics (ROC) curves were constructed for score points and risk categories of CURB-65, CRB-65 and CCI. The area under the ROC curve (AUROC) was calculated with corresponding 95% confidence intervals (CI). As the primary endpoint, we use death from any cause at 1 year after hospital admission for CAP.To calculate the sample size, we used StatsDirect Software . All other analyses were performed with SPSS for Windows .Data of 587 inpatients with CAP were extracted from the hospital´s CAP database. Patients referred from other hospitals (4 patients), those who either discontinued or did not undergo treatment (18 patients) and patients on mechanical ventilation on admission (six patients) were excluded. Of 559 remaining patients, 61 died within 30 days after admission, leaving 498 patients for analysis. Baseline characteristics of the study sample are presented in Table The patients’ median age was 77.7 years . 279 patients were male (56.0%). Functional impairment was prevalent in the study sample, with 111 patients being nursing home residents (22.3%). Comorbid conditions such as congestive heart failure , dementia , and diabetes mellitus  were also frequent. 417 patients (83.7%) scored at least 1 point according to the CCI and 303 individuals (60.8%) had advanced comorbidity. In terms of pneumonia severity, 205 patients (41.2%) were classified into the CURB-65-defined low-risk group, whereas 293 patients (58.8%) were at intermediate or high risk. With CRB-65, 86 patients scored 0 points and hence were classified into the low-risk group.Of 498 patients, 106 21.3%) died within 1 year. Table 1.3% diedROC analysis yielded a weak, yet comparable test performance for CURB-65 and CCI. AUROC values and corresponding CI for score points and risk categories, respectively, were as follows: for CURB-65 0.658 0.606-0.711] and 0.652 [0.598-0.706]; for CRB-65 0.621 [0.565-0.677] and 0.590 [0.533-0.646]; and for CCI 0.647 [0.592-0.702] and 0.631 [0.575-0.688]. The ROC curves for risk categories are shown in Figure 1 and 0.6The main finding of our study is that none of the scores performed satisfactorily in predicting 1-year mortality after inpatient treatment of predominantly older adults with CAP. It is noteworthy, however, that the simple CURB-65 pneumonia severity score appears to be on par with the CCI in this respect. This result may be surprising, given the importance of comorbidity for the long-term outcome of CAP ,15,17-19Contrary to the CCI, the CURB-65 and CRB-65 scores are viewed as markers of disease severity on admission similar to the PSI. Each of the pneumonia severity scores is well validated for predicting 30-day CAP mortality . The simAlthough not satisfying as prognostic tests, the relationship between CURB-65 and CRB-65 and 1-year mortality may be helpful for clinicians, as no other gold standard is available. Compared to more complex tools such as CCI or PSI, the advantages of the CURB-65 and CRB-65 are striking: the scores are easy to calculate on admission even in a busy emergency department; CURB-65 and CRB-65 scores are already implemented in current guidelines for site-of-care decisions ,4; for mThe fact that surveillance needs to be improved has once again been demonstrated with our study. We found that more than one-fifth of patients who survived the index hospitalisation due to CAP died within 1 year after admission. Hsu et al. found that 17% of CAP hospital survivors and 34.4% of hospital survivors of health care-associated pneumonia (HCAP) died within the first year following discharge . Kaplan Apart from CCI, CURB-65 and CRB-65, several other variables were associated with increased 1-year mortality in our sample, including age ≥65 years, nursing home residency, hemiplegia, dementia and congestive heart failure. Nursing home residency and impaired cognitive function have already been described as risk factors for unfavourable long-term outcome in CAP patients, along with congestive heart failure and cardiovascular disease ,27,30,31Several limitations of the current study should be acknowledged. First, we performed a retrospective study, and we used registry data for analysis. However, we do not believe that this affects the validity of our findings. The data in use were mainly assessed and documented during the hospital stay of a given patient. All variables of the pneumonia severity scores were included, with the exception of blood urea levels. As blood urea is part of the standard laboratory measurements obtained for any hospital admission, urea values could be retrieved from the hospital’s information technology system for all cases. The CCI was calculated using ICD-10 codes from administrative data with a well-validated method. Finally, the primary endpoint, death from any cause 1 year after admission, was assessed using data from the city’s local register office, which is reliable even when performed retrospectively.Second, only inpatients with pneumonia were included in our analysis. Current guidelines recommend outpatient treatment for CAP only for CURB-65 or CRB-65-defined low-risk patients and in the absence of advanced comorbidity. It is therefore unlikely that patients from risk groups other than the low-risk group were missed. However, this cannot be verified with our data, and hence our findings cannot be transferred to outpatients with CAP without reservation.Third, findings were made in predominantly elderly patients with high prevalence of comorbid diseases and from a single centre only. Therefore, our findings also need to be validated in other samples and settings.In conclusion, our findings demonstrate substantial 1-year mortality in hospital survivors of CAP. Neither CURB-65 or CRB-65 nor CCI allow excellent discrimination in terms of predicting longer term mortality. Despite its simplicity, CURB-65 is significantly associated with long-term mortality and performed equally to the CCI in this respect. This fact may help to identify patients at higher risk after discharge from hospital. Further research is needed on prognostic markers as well as surveillance and rehabilitation for CAP patients who are considered clinically cured to reduce long-term mortality.Written informed consent was not obtained, because the hospital’s CAP database only contained anonymous data that were used in retrospect. The information on vital status received from the local register office was added by means of another anonymised file using a study specific patient identifier. Detailing this procedure, we applied for ethics approval. The ethics committee of the University of Bochum approved the study without any objection (registration no. 4376-12).